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Oral cancer incidence and mortality are increasing over time. The most common therapies for oral cancers are surgery and radiotherapy, either used alone or combined, and immunotherapy can be also an option. Although there are several therapeutic options, none of them are completely effective, and in addition, there are numerous associated side effects. To overcome these limitations, researchers have been trying to reduce these drawbacks by using drug delivery systems that carry drugs for specific delivery to cancer cells. For that purpose, RNA-coated liposomes to selectively deliver the ligands C 8 (acridine orange derivative) and dexamethasone to oral cancer cells were produced, characterized, and biologically evaluated. Firstly, the RNA structure and binding interaction with ligands (C 8 and dexamethasone) were evaluated by circular dichroism (CD), thermal difference spectroscopy (TDS), nuclear magnetic resonance (NMR) and fluorescence titrations. The biophysical assays evidenced the formation of an RNA hairpin and duplex structure. Moreover, steady-state and time-resolved fluorescence intensity and anisotropy experiments show that C 8 forms a complex with RNA and adopts an open conformation upon RNA binding. Then, RNA-coated liposomes were characterized by dynamic light scattering, and diameters near 160 nm were observed. Time-resolved anisotropy measurements of C 8 loaded in RNA-functionalized liposomes indicate the co-existence of free C 8 in solution (inside the liposome) and C 8 bound to RNA at the external liposome surface. The RNA-functionalized liposomes loaded with C 8 or dexamethasone mediated a significant reduction in the cell viability of malignant UPCI-SCC-154 cells while maintaining viable non-malignant NHDF cells. Additionally, the liposomes were able to internalize the cells, with higher uptake by the malignant cell line. Overall, the results obtained in this work can contribute to the development of new drug delivery systems based on RNA-coated liposomes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Cognitive dysfunction in Parkinson's disease (PD) has received increasing attention, and, together with other non-motor symptoms, exert a significant functional impact in the daily lives of patients. This article aims to compile and briefly summarize selected published data about clinical features, cognitive evaluation, biomarkers, and pathophysiology of PD-related dementia (PDD). The literature search included articles indexed in the MEDLINE/PubMed database, published in English, over the last two decades. Despite significant progress on clinical criteria and cohort studies for PD-mild cognitive impairment (PD-MCI) and PDD, there are still knowledge gaps about its exact molecular and pathological basis. Here we overview the scientific literature on the role of functional circuits, neurotransmitter systems (monoaminergic and cholinergic), basal forebrain, and brainstem nuclei dysfunction in PD-MCI. Correlations between neuroimaging and cerebrospinal fluid (CSF) biomarkers, clinical outcomes, and pathological results are described to aid in uncovering the neurodegeneration pattern in PD-MCI and PDD., (Copyright © 2020. Published by Elsevier B.V.)

Acute and repeated exposure to cocaine alters the cognitive performance of humans and animals. How each administration schedule affects the same memory task has yet to be properly established in nonhuman primates. Therefore, we assessed the performance of marmoset monkeys in a spontaneous object-location (SOL) recognition memory task after acute and repeated exposure to cocaine (COC; 5mg/kg, ip). Two identical neutral stimuli were explored on the 10-min sample trial, after which preferential exploration of the displaced vs the stationary object was analyzed on the 10-min test trial. For the acute treatment, cocaine was given immediately after the sample presentation, and spatial recognition was then tested after a 24-h interval. For the repeated exposure schedule, daily cocaine injections were given on 7 consecutive days. After a 7-day drug-free period, the SOL task was carried out with a 10-min intertrial interval. When given acutely postsample, COC improved the marmosets' recognition memory, whereas it had a detrimental effect after the repeated exposure. Thus, depending on the administration schedule, COC exerted opposing effects on the marmosets' ability to recognize spatial changes. This agrees with recent studies in rodents and the recognition impairment seen in human addicts. Further studies related to the effects of cocaine's acute×prior drug history on the same cognitive domain are warranted., (© 2017 Elsevier B.V. All rights reserved.)

Lately, researchers have made several efforts to improve vaccine production to fight highly contagious respiratory diseases like influenza. One of the most promising options for reducing the impact of this virus is DNA vaccination. However, a large quantity of highly pure plasmid DNA (pDNA) is necessary to attain this goal. The present work describes the production and purification of the plasmid NTC7482-41H-VA2HA expressing influenza virus hemagglutinin using an agmatine monolith. This ligand was chosen to purify supercoiled (sc) pDNA from complex lysates because of its versatile multimodal character. Its natural intervention in several biological systems together with its similarity with the highly studied arginine ligand allowed the development of a simpler and more specific purification process. Agmatine works under two strategies: descending ammonium sulfate gradient and ascending sodium chloride gradient. Furthermore, pH manipulation revealed an important role in pDNA isoforms selectivity. Dynamic binding capacity (DBC) experiments were performed varying different parameters and showed an increase with pDNA concentration, while high flow rate and high pH had the opposite effect. Sc pDNA was purified with high yield and was efficient with respect to cell transfection and cell viability. This monolith showed to be appropriate to purify the plasmid NTC7482-41H-VA2HA, providing a valuable tool for pDNA influenza vaccines preparation., (Copyright © 2016. Published by Elsevier B.V.)

Alternaria alternata is one of the most common saprophytes worldwide that is clinically and epidemiologically associated with severe asthma. Therefore, the identification and characterization of all A. alternata allergens are of major clinical importance. This study describes a new cross-reactive A. alternata allergen that was officially named Alt a 15 by the official Allergen Nomenclature Subcommittee. The complete coding region for Alt a 15 was amplified using 5' and 3' rapid amplification of cDNA ends and PCR. The recombinant protein was produced in Escherichia coli as a 65-kDa fusion protein, and the protein sequence exhibits high homology with several important fungal allergens. Immunoblotting analyses revealed that IgE antibodies from A. alternata-sensitized patients (n=59) bound to rAlt a 15 with a prevalence of 10.2%. All patients who presented sIgE to rAlt a 15 were apparently poly-sensitized to A. alternata and C. lunata. The extensive cross-reactivity between A. alternata and C. lunata serine proteases was confirmed using immunoblotting inhibition assays. Overall, Alt a 15 is an important new cross-reactive allergen of A. alternata that explains some allergies to A. alternata without Alt a 1 sensitization and initial diagnostic errors for allergies to Alternaria. This molecule may improve the accuracy of the diagnosis, the understanding, and the management of IgE-mediated fungal diseases., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

The aromatic ring systems are among the most stable chemical structures known and in combination with many other chemical groups, they can originate an extraordinary variety of molecules, with interesting chemical and physical properties. Many aromatic molecules have been applied for the purification of various biomolecules, such as proteins, carbohydrates and nucleic acids. Combining aromatic chromatography with optimized production, extraction and clarification procedures, can offer a number of advantages for pharmaceutical plasmid DNA (pDNA) purification. This review focuses on pDNA chromatographic purification and analysis using aromatic ligands. The goal is to give an updated view of all existing aromatic ligands, their main characteristics, applicability and technical features of the chromatographic methods in which they have been applied. Also, a critical assessment of each method is performed as well as a comparison of the different procedures, their key features and limitations., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The present study, reports the utilization of berenil as ligand in a negative pseudo-affinity chromatographic step to purify the plasmid pVAX1-LacZ from Escherichia coli clarified lysates. The chromatographic support was prepared by coupling berenil to epoxy-activated Sepharose and was qualitatively and quantitatively characterized using scanning electron microscopy, Fourier transformed infrared spectroscopy and elemental analysis. The clarified lysate was loaded onto the berenil-Sepharose support with 0.55M of ammonium sulphate in the eluent, achieving the immediate elution of plasmid DNA. The impurities tightly bound to the support, were eluted after decreasing the salt concentration to 0M. The overall process enabled the recovery of 87% of loaded plasmid DNA with a HPLC purity of ≫99% and according to FDA specifications. This method represents an alternative approach to the previous utilization of the same chromatographic pseudo-affinity support in a positive mode. It uses lower amounts of salt and one-step chromatographic procedure, resulting in smaller operating time and costs and representing an alternate procedure for plasmid DNA purification., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Affinity chromatography represents a sole technique in purification of different biomolecules. The specific recognition between affinity ligands and target biomolecules has a major role in the specificity of the process. Therefore, choosing the right ligand is a crucial step for the development of a successful purification system. This work describes the application of the DNA intercalator 3,8-diamino-6-phenylphenanthridine (DAPP) as a chromatographic affinity ligand for the specific separation and purification of supercoiled plasmid DNA (pDNA). The support was prepared by coupling DAPP onto an epoxy-activated Sepharose matrix, using mild conditions and resulting in a ligand density of 0.15mmolDAPP/g derivatized Sepharose. The characterization of DAPP-Sepharose support in terms of dynamic binding capacity was achieved by studying the effect of plasmid DNA concentration and flow rate on pDNA adsorption. The maximum capacity value of 336.75μgpDNA/mL gel was obtained at 1mL/min with a pDNA concentration of 150μg/mL. Moreover, the values did not vary significantly with the variation of flow rate. In addition, the DAPP-Sepharose showed a high affinity towards pDNA as quantified by the respective dissociation constant (Kd=2.29±0.195×10(-7)M). The support was also tested for the purification of two plasmid molecules with different sizes (pVAX1-LacZ and pCAMBIA-1303, with 6.05kbp and 12.361kbp, respectively) from clarified Escherichia coli lysate solutions. Total retention of all lysate components was achieved without any added salt to the eluent buffer. The selective elution of impurities and supercoiled pDNA was accomplished simply by the addition of small amounts of salt to the buffer solution. The yield for pCAMBIA-1303 was 65% and for pVAX1-LacZ was 94%, with all host impurity levels in accordance with the requirements established by the regulatory agencies., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Small molecules that bind DNA with high specificity present a promising opportunity for application as chromatographic ligands for plasmid DNA (pDNA) purification. This research used the intercalator 3,8-diamino-6-phenylphenanthridine (DAPP) as an immobilized ligand for the specific separation of supercoiled (sc) pDNA by affinity chromatography. The results showed that the protonated DAPP-Sepharose support has a great affinity for sc pDNA isoform, separating it from the less active open circular and linear isoforms. All pDNA isoforms were retained in the column using 10mM acetate buffer pH 5. Selective elution of oc and linear isoforms was achieved with 0.22M of sodium chloride in the same buffer. Finally, increasing the concentration to 0.55M led to the elution of the sc isoform. The binding of pDNA to DAPP-Sepharose varies in function of pH, and the stability of the protonated DAPP-DNA complex decreases with increasing salt concentration., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Cocaine is a widely abused drug which can result in the establishment of addiction. The neurokinin3-receptor (NK3-R) has been linked to cocaine addiction by genetic, epigenetic, and pharmacological studies suggesting that a cocaine-induced increase in NK3-R signaling may contribute to the establishment of cocaine addiction-related behaviors., Methods: Here we measured cocaine-induced sensitization of vigilance- and locomotor behaviors in marmoset monkeys (Callithrix penicillata) in an open field., Results: We found a sensitization of vigilance-related, but not locomotor behaviors after repeated cocaine (7mg/kg, i.p.) treatment. There was a cross-sensitization for scan frequency, but not of glance frequency, both vigilance-related behaviors, after repeated treatment with the NK3-R agonist senktide (0.2mg/kg, i.p.) given for 7 days, after a cocaine challenge (5mg/kg, i.p.)., Conclusions: These data suggest that in marmoset monkeys, repeated cocaine treatment leads to a sensitization of vigilance-related behaviors, which have a prominent role in spontaneously expressed activities in this species, but not of locomotor activity. Repeated activation of NK3-Rs can mimic some of the behavioral sensitization effect and may thus contribute to the establishment of cocaine related behaviors., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Although cocaine induces several behavioral and hormonal effects, little is known about non-contingent repeated administrations in non-human primates. Therefore, we analyzed behavioral (locomotion, vigilance) and hormonal (cortisol) responses of adult black tufted-ear marmosets during repeated administrations and withdrawal trials. The subjects were divided into two groups (saline or cocaine 5mg/kg, ip) and submitted to nine treatment trials and four withdrawal trials in the absence of any treatment in an open-field arena. Blood samples were obtained on five different time points of the procedure to evaluate the effects of repeated cocaine treatment on basal cortisol levels. Cocaine repeatedly administered to drug-naïve marmosets induced a slow-onset hypervigilance effect (i.e., scan - long-lasting sweeping movements of the head directed at the environment; and glance - single rapid movement of the head directed at the environment), with no concomitant change in locomotion. Treatment cessation during withdrawal immediately reversed the cocaine-induced hypervigilance effect. Cortisol levels remained constant throughout the procedure. Therefore, marmosets seem to have a similar behavioral - but not hormonal - response as humans and other nonhuman primates repeatedly injected with cocaine, but differ from rats in their absence of hyperlocomotor activity. The development of hypervigilance with repeated application may constitute a unique measure to assess cocaine-induced changes in behavior in the marmoset and other nonhuman primates., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In this study, berenil was tested as a ligand, specifically to purify plasmids of different sizes pVAX1-LacZ (6.05 Kbp) and pCAMBIA-1303 (12.361 Kbp) from clarified Escherichia coli alkaline lysates. For this purpose, chromatographic experiments were performed using Sepharose derivatized with berenil. The results showed that both pDNA molecules are completely purified using lower amounts of salt in the eluent than those previously reported for other pseudo-affinity and hydrophobic interaction chromatography based processes. Total retention of all lysate components was achieved with 1.3M ammonium sulphate in the eluent buffer and pDNA elution was obtained by decreasing the salt concentration to 0.55 M. All impurities were eluted after decreasing the concentration to 0M. The recovery yield for pCAMBIA-1303 (45%) was lower than that obtained for pVAX1-LacZ (85%), however the larger pDNA showed a higher purity level. Purification of pVAX1-LacZ was also performed using non-clarified E. coli process streams, replacing the clarification step with a second chromatographic run on the berenil-Sepharose. Using the same binding and elution conditions as before, a pure plasmid sample was obtained with a 33% yield and with all host impurity levels in accordance with the requirements established by the regulatory agencies. These results suggest that this chromatographic method is a promising alternative to purify pDNA for therapeutic use., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The growing demand on plasmid DNA (pDNA) manufacture for therapeutic applications requires a final product with higher quality and quantity, spending the least time. Most of the current processes for pDNA production use at least one chromatographic step, which often constitutes a key-step in the purification sequence. Monolithic stationary phases are new alternatives to the conventional matrices, which offer fast separation of pDNA due to their excellent mass transfer properties and their high binding capacity for large molecules, as pDNA. However, the efficient recovery of pure pDNA focuses on a suitable balance of the feedstock, adsorbent and mobile phase properties. To satisfy the increasing demand for pharmaceutical grade plasmids, we developed a novel downstream process which overcomes the bottlenecks of common lab-scale techniques while complying with all regulatory requirements. This work reports an integrative approach using the carbonyldiimidazole monolith to efficiently purify the supercoiled (sc) pDNA active conformation from other plasmid topologies and Escherichia coli impurities present in a clarified lysate. The monolith specificity and selectivity was also assessed by performing experiments with plasmids of several sizes of 2.7, 6.05 and 7.4 kilo base pairs (kbp), verifying the applicability to purify different plasmids. Hence, the process yield of the pDNA purification step using the CDI monolith was 89%, with an extremely reduced level of impurities (endotoxins and gDNA), which was reflected in good transfection experiments of the sc plasmid DNA sample. Overall, the analytical results and transfection studies performed with the pDNA sample purified with this monolithic enabling technology, confirmed the suitability of this pDNA to be used in pharmaceutical applications., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Small molecules, like some antibiotics and anticancer agents that bind DNA with high specificity, can represent a relevant alternative as ligands in affinity processes for plasmid DNA (pDNA) purification. In the current study, pDNA binding affinities of berberine, berenil, kanamycin, and neomycin were evaluated by a competitive displacement assay with ethidium bromide using a fluorimetric titration technique. The binding between pDNA and ethidium bromide was tested in different buffer conditions, varying the type and the salt concentration, and was performed in both the absence and presence of the studied compounds. The results showed that the minor groove binder berenil has the higher pDNA binding constant. Chromatographic experiments using a derivatized column with berenil as ligand showed a total retention of pDNA using 1.3M ammonium sulfate in eluent buffer. A selective separation of supercoiled and open circular isoforms was achieved by further decreasing the salt concentration to 0.6M and then to 0M. These results suggest a promising application of berenil as ligand for specific purification of pDNA supercoiled isoform by pseudo-affinity chromatography., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The use of therapeutics based on plasmid DNA (pDNA) relies on procedures that efficiently produce and purify the supercoiled (sc) plasmid isoform. Several chromatographic methods have been applied for the sc plasmid purification, but with most of them it is not possible to obtain the required purity degree and the majority of the supports used present low capacity to bind the plasmid molecules. However, the chromatographic monolithic supports are an interesting alternative to conventional supports due to their excellent mass transfer properties and their high binding capacity for pDNA. The separation of pDNA isoforms, using short non-grafted monolithic column with CarbonylDiImidazole (CDI) functional groups, is described in the current work. The effect of different flow rates on plasmid isoforms separation was also verified. Several breakthrough experiments were designed to study the effect of different parameters such as pDNA topology and concentration as well as flow rate on the monolithic support binding capacity. One of the most striking results is related to the specific recognition of the sc isoform by this CDI monolith, without flow rate dependence. Additionally, the binding capacity has been found to be significantly higher for sc plasmid, probably because of its compact structure, being also improved when using feedstock with increased plasmid concentrations and decreased linear velocity. In fact, this new monolithic support arises as a powerful instrument on the sc pDNA purification for further clinical applications., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Cholesterol and its precursors, namely 7-dehydrocholesterol, desmosterol and lathosterol are important biochemical markers of cholesterol biosynthesis, and their quantification in body fluids is useful for the diagnosis of cholesterol biosynthesis pathway disorders. A rapid and sensitive gas chromatographic-mass spectrometric method was developed and validated for quantitative analysis of five sterols (cholesterol, 7-dehydrocholesterol, desmosterol, lathosterol and sitosterol) in amniotic fluid. The method was linear for all compounds (r(2)>0.99), and intra and inter-assay coefficients of variation were typically below 5%, and inaccuracy was within a +/-12% interval. The method was applied to 330 amniotic fluid samples, grouped by gestational age between 13 and 22 weeks of pregnancy, in order to establish reference intervals for sterols in this specimen. The obtained concentrations (mumol/L) for each sterol was as follows: 22.1758+/-4.2716 at 13 weeks and 78.5082+/-12.9041 at 22 weeks for cholesterol; 0.0039+/-0.0007 at 13 weeks and 0.1150+/-0.0212 at 22 weeks for 7-dehydrocholesterol; 0.1562+/-0.0406 at 13 weeks and 0.7691+/-0.0821 at 22 weeks for desmosterol; 0.0272+/-0.0035 at 13 weeks and 0.8551+/-0.1791 at 22 weeks for lathosterol; and 0.0404+/-0.0039 at 13 weeks and 0.2326+/-0.0386 at 22 weeks for sitosterol. The method was also applied to one pathological sample that showed decreased levels of cholesterol, and higher concentration of 7-dehydrocholesterol, which is consistent with a 7-dehydrocholesterol-reductase deficiency. Our results showed that as long as pregnancy goes on, the concentrations of cholesterol and precursors increase in amniotic fluid, which is related to the increased need for cholesterol by the fetus. The reference range of each sterol in amniotic fluid was calculated at different gestational ages and will be useful for the interpretation and validation of biochemical prenatal diagnosis of inborn errors of sterol biosynthesis., (Copyright 2010 Elsevier B.V. All rights reserved.)

Initial investigations indicated the use of the Marmoset Predator Confrontation Test (MPCT) as an experimental procedure to measure fear/anxiety-related behaviors in non-human primates. However, possible long-term habituation effects and re-use of experimental subjects need to be verified. This study, therefore, compared the behavioral response of experienced versus naïve adult black tufted-ear marmosets (Callithrix penicillata) in the MPCT, with/without diazepam administrations. Subjects were tested in the figure-8 maze and confronted with a taxidermized wild-cat predator stimulus. After four initial 20-min maze habituation sessions, each subject was submitted to two randomly-assigned 20-min predator confrontation sessions: vehicle and 2 mg/kg of diazepam. Confrontation with the predator induced significant behavioral changes; i.e., proximic avoidance and tsik-tsik alarm call. Diazepam administration, concomitant to predator exposure, reversed the behavioral changes observed. In both the experienced and naïve marmosets a similar behavioral profile and response pattern to diazepam was detected, corroborating the important selective pressure that felines seem to have on marmoset behavioral ecology. Therefore, during a more naturalistic-like regimen--i.e., recurring intermittent predator encounters--the general response pattern remains highly consistent, regardless of prior experience. One may consider the re-use of marmoset subjects in the MPCT, particularly under these specific conditions (i.e. repeated 20-min confrontations, 72-h apart).

A detailed behavioral analysis of nine Callithrix penicillata was conducted in the Marmoset Predator Confrontation Test (MPCT) during (a) four habituation trials with no "predator," (b) six confrontation trials with the predator (taxidermized oncilla cat, Felis tigrina), and (c) four trials with the predator removed. The marmosets habituated to the test apparatus with significant decreases in locomotion, exploration and long calls. Initial exposure to the predator elicited mainly fear-related behaviors (proximic avoidance, tsik-tsik vocalization, swaying/tongue in-out), whereas repeated confrontations attenuated these behaviors, concomitant to an increase in anxiety-associated responses (scratching/grooming/scent marking). The initial behavioral repertoire, observed before confrontations, was fully restored only upon removal of the predator. This easily discernable complex defensive behavioral repertoire is hoped to provide a comprehensive baseline for studying the biological substrates of fear/anxiety parameters in nonhuman primates.

In order to further validate the recently developed marmoset (Callithrix penicillata) predator confrontation model of fear and anxiety, we investigated the behavioral effects of buspirone with this method. The apparatus consisted of three parallel arms connected at each end to a perpendicular arm, forming a figure-eight continuous maze. A taxidermized wild oncilla cat (Felis tigrina) was positioned facing a corner of the parallel arms, alternating between the left or right side of the maze among animals tested. All subjects were first submitted to seven 30-min maze habituation trials (HTs) in the absence of the predator, and then to five randomly assigned treatment trials (TTs) in the presence of the predator: three buspirone sessions (0.1, 0.5 and 1.0 mg/kg), saline and sham injection controls. Twenty minutes after treatment administration, the animal was released into the maze and had free access to the apparatus for 30 min. All trials were taped for later behavioral analysis. Buspirone significantly decreased the frequency of scent marking, while increasing the time spent in proximity to the 'predator' stimulus, indicating an anxiolytic effect. Neither locomotor activity, exposure to a novel environment, stimulus location and habituation, nor gender influenced the effects of the drug treatments. These results further validate this method and demonstrate the potential usefulness of this ethologically based paradigm to test anxiety and fear-induced avoidance in nonhuman primates and its susceptibility to anxiolytic pharmacological manipulations.

The neurokinin Substance P (SP) is widely distributed in the central nervous system and has been extensively studied in various functional aspects. This review focuses on the behavioral relevance of SP. Here we show that SP can have memory-promoting, reinforcing and anxiolytic-like effects when administered systemically or into the nucleus basalis of the ventral pallidum. These effects seem to be mediated via the SP-preferring NK(1)receptor and differentially related to N- versus C-terminal fragments of the undecapeptide. Secondly, SP injection into the ventral pallidum can lead to increases of acetylcholine in frontal cortex and dopamine in nucleus accumbens, suggesting that the hypermnestic, positively reinforcing and anxiolytic effects observed upon basal forebrain injection of SP are mediated by activation of the nucleus accumbens-ventral pallidum circuitry. Furthermore, SP and certain SP-fragments may not only be considered to have beneficial behavioral effects in normal animals, but can also prevent lesion-induced functional deficits and improve the speed of recovery. This indicates that SP agonists might also have a neuroprotective capacity in parallel with recovery-promoting actions., (Copyright 2000 Harcourt Publishers Ltd.)

This work reports new studies on cellulases fractionation by hydrophobic interaction chromatography. The purification procedure for the Trichoderma reesei cellulase complex consists of gel permeation chromatography on Sephadex G-25M followed by an ultrafiltration step. The concentrated enzyme solution was then fractionated on Sepharose CL-6B modified by covalent immobilization of 1,4-butanediol diglycidyl ether. The influence of the mobile phase composition on the chromatographic behaviour of the T. reesei cellulase complex was investigated. By using 13% (w/v) ammonium sulphate in eluent buffer, a selective separation of beta-glucosidase with a two-fold increase in specific activity and a recovery of 60% cellobiase activity were obtained. Other commercial hydrophobic supports (octyl- and phenyl-Sepharose) were also tested and compared under the same conditions.

The elevated T-maze, an ethologically based test, has been used to investigate the effects of anxiolytic drugs on memory and the relationships between neural systems involved in such modulation. This test allows the measurement in the same rat of two kinds of aversively motivated behaviors--inhibitory avoidance and one-way escape. The apparatus consists of three arms of equal dimensions, elevated 50 cm from the floor. One arm is enclosed by walls and stands perpendicular to the two open arms. Placing the rat at the end of the enclosed arm and recording the time to withdraw from this arm during three consecutive trials assesses inhibitory avoidance. Soon afterwards, the rat is placed at the end of one of the open arms and the time to leave this arm recorded as escape response. Three days later memory is assessed by reexposing the rats to the maze. One critical question raised by these studies is whether the anterograde amnesia induced by anxiolytic drugs could be due to insufficient learning during training or to amnesia. The present work investigated whether the introduction of a multitrial training-to-criterion procedure could overcome this question. For this purpose, rats were tested as many times as needed to stay in the enclosed arm continuously for 300 s (avoidance learning to criterion). Results from Experiment 1 showed that rats trained to a learning criterion shows significantly better retention performance. Experiment 2 evaluated the effects of pretraining diazepam (DZP) treatment on this training-to-criterion protocol. The results indicate that DZP did not affect acquisition performance but induced a dose-dependent impairment of the inhibitory avoidance in the memory test. One-way escape (latency to enter the enclosed compartment from the open arms) was not affected by DZP. These results rule out the possibility that the impairment of inhibitory avoidance memory in the elevated T-maze could be due to lack of learning during training, and support the hypothesis that the disruptive effects of DZP are on processes involved in long-term storage of information.

L-DOPA can often induce psychotic reactions during treatment for Parkinson's disease. This study was undertaken to assess, in an animal model of Parkinson's disease, the impact of L-DOPA treatment on two potential biological risk factors for psychosis, namely, an increase in prefrontal cortex dopamine and an increase in the stress-related hormone corticosterone. Hemiparkinsonian rats with unilateral 6-hydroxydopamine (6-OHDA) lesions which resulted in severe unilateral denervation of dopamine neurons were treated with either saline or 25 mg/kg L-DOPA methyl ester (with 2 mg/kg carbidopa). Serum L-DOPA concentrations were found to be positively and highly correlated with serum corticosterone, with medial prefrontal cortex dopamine and with the dopamine metabolite homovanillic acid. Serum L-DOPA, however, was found not to be correlated with serum or brain concentrations of serotonin, 5-hydroxyindoleacetic acid, or norepinephrine. These findings support the possibility that chronic L-DOPA treatment can expose parkinsonian patients to two significant risk factors for psychosis: 1) increased levels of prefrontal cortex dopamine, and 2) increased levels of serum corticosterone.

In an attempt to analyze different types of anxiety, and at the same time assess memory, a new experimental model was developed. The apparatus, named the elevated T-maze, consisted of three arms of equal dimensions (50 x 10 cm) elevated 50 cm from the ground. One arm, enclosed by 40-cm high walls, was perpendicular to two open arms. The first experimental session was conducted 25 min after IP injection of either drug or saline. To assess inhibitory (passive) avoidance, the rat was placed at the end of the enclosed arm and the time taken to withdraw from this arm was recorded three times in succession. Soon afterwards, the rat was placed at the end of one of the open arms and the time taken to withdraw from this arm was measured, thus estimating one-way escape. To assess memory, inhibitory avoidance and escape were measured again 3 days later, without drug. Dose-response curves were determined for the benzodiazepine anxiolytic and amnestic agent diazepam (DZP, 0.5-4 mg/kg), as well as for ipsapirone (IPS, 0.25-2 mg/kg), an azapirone anxiolytic that is devoid of clinically significant amnestic effects. The doses of 1, 2, and 4 mg/kg DZP and of 1 and 2 mg/kg IPS impaired inhibitory avoidance, an effect that may be viewed as anxiolytic. Inhibitory avoidance remained impaired 3 days later in the rats treated with 1-4 mg/kg DZP, indicating anterograde amnesia. This effect was not due to state-dependent learning, because rats injected both at pretraining and pretesting with 2 mg/kg DZP still showed complete amnesia.(ABSTRACT TRUNCATED AT 250 WORDS)

Rats with severe unilateral dopamine denervation (> or = 95% dopamine deficit), produced by intracerebral injection of 6-hydroxydopamine (6-OHDA) into the ventral tegmentum nigrostriatal dopamine neurons, were administered 25 mg/kg L-dopa (3,4-dihydroxyphenylalanine) methyl ester/2 mg/kg carbidopa. The neurochemical effects of the L-dopa treatment on uric acid in the cortex and striatum of the intact and 6-OHDA hemisphere were measured. In comparison to saline animals, uric acid concentrations in brain were increased in the L-dopa treated animals. There were no interhemispheric differences in the uric acid concentrations either in the L-dopa or in the saline treated animals. Interhemispheric differences were, however, observed in terms of the correlations obtained between L-dopa and uric acid concentrations in the intact vs. the 6-OHDA hemisphere. Statistically significant correlation coefficients were found in the striatal and cortex samples obtained from the 6-OHDA hemisphere. Furthermore, high correlation coefficients were observed between contralateral rotation frequencies and uric acid concentrations in the cortex and striatum of the 6-OHDA hemisphere. In contrast, only low and statistically non significant correlations were observed in the tissue samples obtained from the intact hemisphere. These observations suggest that L-dopa activation of the dopamine supersensitive receptors of the DA denervated hemisphere and the associated metabolism of purines with high energy phosphate bonds (e.g. ATP and GTP) increases uric acid as an end-product of purine metabolism. These findings are consistent with other findings indicating that uric acid in the brain can provide an index of metabolic activation in brain tissue.

In order to test the effects of sodium barbitone on the acquisition and retention of an appetitively and an aversively reinforced behavior, mice were trained in a spatial discrimination Y-maze task. Learning was observed in both situations, with acquisition unimpaired by the drug. Sodium barbitone did, however, affect retention of both tasks in all groups treated with the drug before training. Results are discussed in light of the various modes of action of this drug, i.e., as an inhibitor of protein synthesis, as a blocker of catecholamine biosynthesis, with regard to its effects on paradoxical sleep and on gamma-amino-butyric acid (GABA).

Experiments were undertaken to investigate the effects of the neuropeptide Substance P (SP) on performance of a conditioned inhibitory avoidance response in rats. A single-trial inhibitory avoidance task was employed. In Experiment 1 SP was injected IP immediately after the training trial in doses of 0.5, 5, 50, 100, 250 or 500 micrograms/kg; control animals were injected with diluent vehicle. The group treated with 50 micrograms SP/kg exhibited better avoidance than the other groups. In Experiment 2 the doses of SP used were 1, 50, 250 micrograms/kg, and the control animals were injected with vehicle or not injected at all. Only the 50 micrograms SP/kg treatment group showed significantly better performance. In Experiment 3 50 micrograms/kg SP or vehicle was injected post-trial immediately or 5 hr after the trial. Only the group in which SP was injected immediately after the training trial showed significantly better performance when tested 24 hr later. This result rules out the possibility that SP exerts its effect by a long lasting proactive action on performance during the testing trial 24 hr later.

Behavioral effects of unilateral microinjections into periventricular structures of bicuculline, a classic GABA-A antagonist, and semicarbazide, a glutamic acid decarboxylase blocker, were studied in detelencephalated rats. These drugs produced a behavioral activation together with jumps. However, the characteristics of this behavioral activation differed as the injections were made in dorsal periaqueductal gray matter or medial hypothalamus. These data show close similarities to those observed with intact animals suggesting that GABA-A receptors are involved in the neural control of expression of flight behavior and functions in an intact manner and possibly independent of influences from forebrain structures. At variance with intact animals, these drugs produced contralateral turning behavior when locally injected into MH, pointing to some kind of inhibitory control exerted by telencephalic structures on the expression of circling behavior from diencephalic regions.